1 00:00:00,004 --> 00:00:04,894 Click Open project and open the saved Figure project. 2 00:00:04,894 --> 00:00:14,121 We will measure the 30 kDa GSDMD signal on the lower half of the membrane, using HSP90 on the upper half as the loading control. 3 00:00:14,121 --> 00:00:19,385 Return to the GSDMD chemiluminescence image. 4 00:00:19,385 --> 00:00:26,495 Drag a horizontal selection around the lower, 30 kDa signal, covering all six lanes. 5 00:00:26,495 --> 00:00:33,065 Adjust the top and bottom edges to include this row of bands and a little surrounding background. 6 00:00:33,065 --> 00:00:38,610 Name this cropped band strip GSDMD 30 kDa. 7 00:00:38,610 --> 00:00:41,817 Click Save strip. 8 00:00:41,817 --> 00:00:50,713 The new strip contains only the 30 kDa signal. The earlier crop with both signals is still in the strip library. 9 00:00:50,713 --> 00:00:56,778 Open Figure builder, then click New figure to create a Figure for quantification. 10 00:00:56,778 --> 00:01:03,302 Name this Figure GSDMD 30 kDa and HSP90 quantification. 11 00:01:03,302 --> 00:01:06,630 Set Lanes to six. 12 00:01:06,630 --> 00:01:12,742 Drag GSDMD 30 kDa from the strip library into the Figure. 13 00:01:12,742 --> 00:01:17,453 Then drag in HSP90 from the same membrane. 14 00:01:17,453 --> 00:01:24,188 Enter the six sample names in matching order: three Control samples, followed by three Drug samples. 15 00:01:24,188 --> 00:01:29,671 The target and loading control in each column represent the same sample. 16 00:01:29,671 --> 00:01:33,259 Open Protein presets. 17 00:01:33,259 --> 00:01:37,533 Enter HSP90 as the protein name. 18 00:01:37,533 --> 00:01:41,582 Enter 90 as the molecular weight. 19 00:01:41,582 --> 00:01:45,072 Select Loading control. 20 00:01:45,072 --> 00:01:48,189 Click Save entry. 21 00:01:48,189 --> 00:01:51,607 Click Densitometry. 22 00:01:51,607 --> 00:01:56,673 Choose Target / same-membrane control for normalization. 23 00:01:56,673 --> 00:02:03,056 Select HSP90 as the loading control for GSDMD 30 kDa. 24 00:02:03,056 --> 00:02:11,236 These strips come from the upper and lower halves of the same membrane. Their six lanes are in the same sample order. 25 00:02:11,236 --> 00:02:16,173 For band polarity, choose Dark bands on light background. 26 00:02:16,173 --> 00:02:25,532 Each lane box defines the measurement region. The software estimates background within the box and sums the background-corrected signal. 27 00:02:25,532 --> 00:02:32,221 First, turn off Equal lane widths and Equal box heights so you can adjust the boxes individually. 28 00:02:32,221 --> 00:02:37,081 Drag the middle boundary to keep neighboring lanes separate. 29 00:02:37,081 --> 00:02:42,278 Select Equal lane widths to make all six boxes the same width. 30 00:02:42,278 --> 00:02:48,021 Move one lane box vertically to align it with the 30 kDa signal. 31 00:02:48,021 --> 00:02:52,525 Drag the blue edge to adjust the height of this box. 32 00:02:52,525 --> 00:02:57,791 Select Equal box heights to give all six boxes the same height. 33 00:02:57,791 --> 00:03:06,992 Check each lane. Include its signal and a little background, keeping the upper, 53 kDa signal outside the boxes. 34 00:03:06,992 --> 00:03:11,759 Now look at the six HSP90 lane boxes. 35 00:03:11,759 --> 00:03:16,751 Check that Equal lane widths is selected for HSP90. 36 00:03:16,751 --> 00:03:21,940 Keep Equal box heights selected for HSP90 as well. 37 00:03:21,940 --> 00:03:30,292 The loading control is at a different vertical position. Check both sets of boxes against the same sample order. 38 00:03:30,292 --> 00:03:35,179 For GSDMD, click Confirm lane regions. 39 00:03:35,179 --> 00:03:40,388 For HSP90, click Confirm lane regions as well. 40 00:03:40,388 --> 00:03:47,962 Click Calculate ratios to measure the original image pixels and calculate background-corrected integrated density. 41 00:03:47,962 --> 00:03:54,997 The result table now lists the target signal, loading-control signal, and ratio for all six lanes. 42 00:03:54,997 --> 00:04:04,180 For each lane, the ratio is the integrated density of GSDMD 30 kDa divided by that of the corresponding HSP90 lane. 43 00:04:04,180 --> 00:04:14,429 Check the six sample names, loading-control values, and background before exporting. These ratios have not been rescaled to a control-group mean of one. 44 00:04:14,429 --> 00:04:20,186 Click Export Excel to save the six lane results and their selection records. 45 00:04:20,186 --> 00:04:25,517 Open the exported Excel workbook to view the results in English. 46 00:04:25,517 --> 00:04:33,231 Each lane includes the target and loading-control integrated densities, their ratio, and the saved ROI coordinates. 47 00:04:33,231 --> 00:04:36,743 Return to the software. 48 00:04:36,743 --> 00:04:43,187 Click Back up project to save the crop, lane regions, and loading-control settings together. 49 00:04:43,187 --> 00:04:51,291 When you reopen this project, you can check the original images and lane boxes, then recalculate the results you need.